effipulp - rahn-group
TRANSCRIPT
2 Cosmetic Active Ingredients
Ppt Effipulp® ENG
January 2012
1. Presentation of Effipulp®
2. The issue of aging
3. Biological activity
• Restoring natural defenses and detoxification systems
• Moisture redensification & strengthening of the stratum corneum
4. Clinical study: Plumping effect
Table of contents
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Ppt Effipulp® ENG
January 2012
• Avocado (Persea Gratissima), mainly Fuerte and Haas varieties, collected in Africa and Latin America
• Family: Lauraceae (laurels), subfamily: Persea (ex. bay laurel, cinnamon, camphor)
• Climacteric fruit, ripening when the fruit is picked
• Native to Mexico, consumed 10,000 years ago by the Aztecs and Mayas, then imported to Europe in the 17th century
Avocado: presentation & history
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Ppt Effipulp® ENG
January 2012
Avocado: Sourcing and Use based on CSR
• Laboratoires Expanscience is confirmed as Trading member of UEBT* since end Dec. 2011
*Union for Ethical BioTrade (http://www.ethicalbiotrade.org/)
• Exemple: top-grafting of old Avocado trees with young ones:
- work on existing cultivated lands (avoid deforestation)
- get back younger trees, able to provide fruits
• Total exploitation of Avocado fruit
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Ppt Effipulp® ENG
January 2012
Avocado: a total exploitation of the fruit…
Pressure
Crude Oil
Molecular Distillation
Unsaponifiables pharmaceutical application (API)
Sliding & drying
Avocado Oil HAREF
Avocado cakes
Esterification
5 a Avocuta®
Hydrolysis
Effipulp®
Avocado powder Fresh fruit
composition:
– 70% water
– 21% lipids
– 4% glucides
– 2% proteins
– 2% fibers
– 1% ashes
Refining
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Ppt Effipulp® ENG
January 2012
… and benefits of a pharmaceutical supply
• Specific preparation of Avocados (included in a patent):
1. water eliminated through drying under controlled conditions
2. oil obtained by pressure
• Unique quality of Avocado “cakes”:
- preserved thanks to the preparation method
- concentrated in proteins
• Laboratoires Expanscience unique supplier of Avocado peptides.
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January 2012
Green process without organic solvent
> Enzymatic hydrolisis of delipidated pressed Avocados (Cellulase – Protease) to obtain peptides
> Purification through ultrafiltration (15 kDa) and nanofiltration (1 kDa)
> Bleaching
> Addition of organic maltodextrin (50%) & freeze-drying
Process respecting the naturalness of the fruit
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January 2012
• No residual proteins (> 10 kDa)
• Reproductibility of the composition in peptides (distribution in molecular weight)
HPTLC chromatography
Characteristics Beige powder with characteristic odour
Protide content (Kjeldahl method) ≥ 15%
Amino a nitrogen content (UV spectrophotometry)
4.0-8.0 g/100 g
Maltodextrin 45.0-55.0 g/100 g
Total micro-organism count
Yeast and mould count
Pathogens
<100 CFU/g
<100 CFU/g
None
Analytical specifications
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Ppt Effipulp® ENG
January 2012
• Shelf life: 3 years
• Packaging: 100 g and 1 kg
• INCI name: Hydrolyzed Avocado Protein and Maltodextrin
• CAS numbers: 1085861-52-4 ; 9050-36-6
• REACH status: Pre-registered
• Patent ref.: WO 2005/105123 - WO 2010/052312
Technical and regulatory data / patents
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Ppt Effipulp® ENG
January 2012
Production of Reactive Oxygen Species (ROS)
• Endogenous: cellular metabolism (cellular respiration)
• Exogenous: UV radiation, stress, pollution etc.
Antioxidant defenses
• Enzymatic: SOD, catalase, glutathion peroxidase
• General: Vit. E, vit. C, vit. A
Systems repairing and eliminating oxidized protein
• Repair: thioredoxin, glutaredoxin etc.
• Elimination (cytosolic): Proteasome
Main factors of skin aging
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Ppt Effipulp® ENG
January 2012
Young skin: Homeostasis between ROS production and natural defenses
Natural defenses
Endogenous and exogenous
production
ROS
ROS
Antioxidant
Repair and elimination systems
Antioxidant
Repair and elimination systems
Main factors of skin aging
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January 2012
Natural defenses
Endogenous and exogenous
production
Mature, weakened skin: Imbalance in homeostasis, cellular damage
Main factors of skin aging
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January 2012
Cellular functions:
• Proliferation and differentiation of keratinocytes and fibroblasts
• Syntheses (HA, collagens, elastin,…)
• Protection systems (proteasome, thioredoxin,…)
ROS
Oxidation
PROTEINS
DNA mutation
Peroxidation
LIPIDS
Str
ess
ROS consequences and skin aging
CONSEQUENCES
Epidermis: alteration of the protective barrier and therefore of the moisture balance
=> dehydrated and stressed skin: loss of moisture, volume
Dermis: alteration of the extra-cellular matrix => mature, weakened skin:
loss of suppleness, firmness and radiance
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Ppt Effipulp® ENG
January 2012
• Enzymatic systems repairing oxidized protein (reduction): • Thioredoxin / Thioredoxin Reductase
• Glutaredoxin
• Methionine Sulfoxide Reductases
• Intracellular proteolytic deterioration The proteasome, potential recycling of amino acids
Sub-units Catalytic activities
Systems repairing and eliminating oxidized protein
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Ppt Effipulp® ENG
January 2012
• Human dermal fibroblasts, treated with 0.005% Avocado peptides over 48 h
• Gene expression analysis by RT-PCR
Activity on thioredoxin - repair system
0,005 %
Avocado peptides
0,01% Effipulp®
*** p<0.001
relative quantity of the gene expression
(normalized)
0 ,0 0
0 ,5 0
1 ,0 0
1 ,5 0
2 ,0 0
2 ,5 0
3 ,0 0
C o n t r o l A v o c a d o p e p t i d e s
0 , 0 0 5 % A I
Qu
an
tit
é R
ela
tiv
e n
or
ma
lis
ée
p
ar
Y
WH
AZ + 1 7 7 %
* * *
0 ,0 0
0 ,5 0
1 ,0 0
1 ,5 0
2 ,0 0
2 ,5 0
3 ,0 0
C o n t r o l A v o c a d o p e p t i d e s
0 , 0 0 5 % A I
Qu
an
tit
é R
ela
tiv
e n
or
ma
lis
ée
p
ar
Y
WH
AZ + 1 7 7 %
* * *
non-treated cells Avocado peptides 0,005%
Effipulp® stimulates the gene expression of thioredoxin
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Ppt Effipulp® ENG
January 2012
• “Senescent” fibroblasts (successive replications) treated over 1 hour and 45 minutes with 0.005% Avocado peptides, or by PA28 (proteasome activator)
• Measurement of enzymatic activity of proteasome (luminescent substrate)
Senescence slows the proteasome’s activity as well as the gene expression
of the sub-unit1
Effipulp® restores the proteasome’s activity as well as the gene expression
of the sub-unit1
Activity on the Proteasome
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
1 4 0
1 6 0
" Y o u n g c e l l s " " S e n e c e n t
c e l l s "
R é f é re n c e
( P A 2 8 a c t i v a t o r
4 n g / m l )
A P 0 , 0 0 5 % A I
Ac
tiv
ité
(%
du
té
mo
in)
+38%
° ***
+43%
°
-32%
° vs senescents cells
*** p<0.001
pro
teasom
e a
ctivity (
%)
"Young cells" "Senescent cells"
Ref. PA28 (4 ng/mL)
Avocado peptides 0,005%
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January 2012
• “Senescent” fibroblasts (successive replications) treated over 72 hours with 0.005% Avocado peptides, or with BHA at 150 µM
• Measurement of the quantity of oxidized proteins (in fibroblasts) using a specific fluorescent probe
The rate of protein oxidation increases
with senescence
Effipulp® inhibits the accumulation
of oxidized proteins
Activity on oxidized proteins
0
5 0 0 0 0
1 0 0 0 0 0
1 5 0 0 0 0
2 0 0 0 0 0
2 5 0 0 0 0
3 0 0 0 0 0
"Y o un g ce l l s " "Se n e s ce n t
ce l l s "
R e fe re nce
(B H A 1 5 0 µM )
A P 0 ,0 0 5% A I* p<0.05
-57%
° *
+35%
-44%
° *
Oxydiz
ed p
rote
ins
(flu
ore
scence inte
nsity)
° vs senescents cells
"Young cells" "Senescent cells"
Ref. BHA (150 µM)
Avocado peptides 0,005%
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January 2012
Protective effect against lipid peroxidation
P=50%
P=64%
Quantitity of lipidic peroxydes
Effipulp® modulates lipid peroxidation and therefore protects the integrity of cellular membranes
• Jurkat cells (cellular model) subject to H2O2 stress and treated with 0.05% Avocado peptides, or with BHA (anti-oxidant ref.) over 30 minutes
• Measurement of the peroxidation of membrane lipids using a specific fluorescent probe (C11-fluor, flow cytometry)
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Ppt Effipulp® ENG
January 2012
Restoration of cellular functions for:
Hygroscopic molecules and proteins syntheses
Cohesion of the stratum corneum
Restoring natural defenses & detoxification systems
Protective effect of Effipulp®
oxidized proteins
Antioxidant effect of Effipulp®
peroxidized lipids
• Repair by thioredoxin
• Elimination by proteasome Cellular membranes protection
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Ppt Effipulp® ENG
January 2012
• Moisture content with age
GAGs and in particular in hyaluronic acid guaranty
a natural hydration of the dermis and epidermis, returns density, elasticity and comfort (trapping of water, up to 1,000 times its weight)
=> Aging: GAGs HA = significant reduction in the skin’s volume (by age 50, half of the HA has disappeared)
• Stratum corneum
The cohesion of the SC limits imperceptible water loss and therefore maintains the moisture level, in particular in the epidermis.
=> Aging: disruption of SC
Hyaluronic acid
Water
Aging: moisture balance and maintenance
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January 2012
Epidermal moisture redensification
0
2 0 0 0
4 0 0 0
6 0 0 0
8 0 0 0
1 0 0 0 0
1 2 0 0 0
1 4 0 0 0
C o n t r o l R e t o in o ic a c id
0 , 1 µ M
A v o c a d o
P e p t id e s
0 , 0 0 5 % ( A I )
* p<0.05 ** p<0.01
+146%
+31%
*
**
HA q
uantity
(n
g/m
l)
Avocado peptides 0,005%
Non treated cells Ref. Retinoic acid (0,1 µM)
Effipulp® stimulates the release of Hyaluronic Acid by keratinocytes
• Normal human keratinocytes treated over 72 hours with 0.005% Avocado peptides, or with retinoic acid at 0.1 µM (ref.)
• Measurement of the release of Hyaluronic Acid (ELISA) in cell culture supernatants
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January 2012
Dermal moisture redensification
0
0 ,5
1
1 ,5
2
2 ,5
3
C o n t ro l "S e n e s c e n t C e lls " A v o c a d o p e p t id e s
0 ,0 5 % ( A I)
*** p<0.001 ** p<0.01
HA q
uantity
(µ
g/m
l)
-64%
+91% vs senescent cells
**
***
"Young cells" "Senescent cells" Avocado peptides 0,05%
• Normal human fibroblasts “senescent” aged with H2O2 treated over 8 days with 0.05% Avocado peptides,
• Measurement of the release of Hyaluronic Acid (ELISA) in cell culture supernatants
Effipulp® stimulates the release of Hyaluronic Acid by fibroblasts under senescent conditions
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Ppt Effipulp® ENG
January 2012
• Topical application of an emulsion with 0.6% Effipulp® vs. placebo on normal skin explants over 72 hours
• Immunostaining of Hyaluronic Acid (green) & image analysis quantification
Effipulp® stimulates the synthesis of Hyaluronic Acid
Epidermis
Dermis
Skin moisture redensification: ex-vivo confirmation
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Ppt Effipulp® ENG
January 2012
• Topical application of an emulsion with Effipulp® (0.6%) vs. placebo on delipidated skin explants (model for dry skin) over 3 hours
• Marking of Filaggrine (SC cohesion, precursor of NMF) and Transglutaminase (corneal envelope) by immunofluorescence (green)
Placebo Effipulp® 0.6% Placebo Effipulp® 0.6%
Effipulp® stimulates the synthesis of molecules involved in the cohesion & formation of the stratum corneum
Corneal layer strengthening: epidermal cohesion
Filaggrine (FG) immunostaining Transglutaminase (TG) immunostaining
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January 2012
• Synthesis of hygroscopic molecules
• Hyaluronic acid in the epidermis and dermis
• GAGs (+34%, in-vitro, keratynocytes, 0.005% AI)
Moisture redensification
• Strengthening of the corneal layer
• Filaggrine, transglutaminase
• Ceramides (+30%), cholesterol (+12%)
(reconstructed epidermis, 0,005% AI)
Preservation of moisture content
Redensification & preservation of moisture content
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January 2012
Stratum Corneum
Granulosum
Spinosum
Dermis
Epidermis
• Innovative and precise technique
> Specific optical measurement for the epidermis (infrared laser, very high definition images)
> 2 acquisition of 400 joint pictures over a distance of 1,6 mm on the same area
> Epidermis thickness and volume quantification (SC excluded)
OCT technique / data by age range
Neerken, S. et al, Journal of Biomedical Optics, 2004
• Bibliographical study comparing the thinning of the epidermis during aging between 2 groups (15 pers. each)
8 0 %
8 5 %
9 0 %
9 5 %
1 0 0 %
Y o u n g
( 2 2 , 5 y e a r s )
A g e d
( 5 5 , 3 y e a r s )
Epidermis thickness evolution by aging
-8.4%
(22,5 years) (55,3 years)
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January 2012
• Comparative study, double-blind vs. placebo
• Effipulp® tested at 0.6% (face cream)
• 49 women - 2 groups of 25 subjects
Thickness values for both groups are comparable with T0
• Inclusion criteria:
age 40-56, average age: 49
dry and sensitive skin types (stinging test)
• Term: 56 days – 2 applications/day on the face
• Measurement (cheeks) of the variation in thickness in relation to the area, or the volume of the epidermis
Clinical study: protocol
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January 2012
> Placebo: no significant difference between D0 and D56 (+1,9%)
> Cream with Effipulp®: +6.1% significant increase (average) in epidermis volume after 56 days
Results
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January 2012
Results: 3D visualisation
Pores or hair-sebaceous structure
Epidermis thickness
Skin surface (below SC)
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January 2012
Activity summary
Protective and
antioxidant effect
• Oxidized proteins - Thioredoxin and Proteasome
• Peroxidized lipids for cellular membranes integrity
Restoration of cellular
functions
•Synthesis of Hyaluronic Acid - Dermis & Epidermis
•Protein synthesis - Cohesion of stratum corneum
Plumping and redensifying
effect
•Increase in the volume of the epidermis
•Strengthening of the stratum corneum for moisture preservation
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Ppt Effipulp® ENG
January 2012
• ANTI-AGING PLUMPING AND DETOXIFYING
• Strengthening the natural defense and detoxification systems - the Proteasome - to restore cellular functions
• Plumping the epidermis and dermis by stimulating the synthesis of hyaluronic acid
• Strengthening the stratum corneum to preserve moisture
• Applications: Face, lips, chest and hands
• Restores lost volume to rejuvenate skin
• Returns firmness and elasticity to the skin
• From the first signs of aging to mature, weakened, dehydrated and stressed skin
• Can complement esthetic cosmetic procedures
• Recommended dose: 0.1% to 0.6%
Conclusion
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January 2012
To find out more…
Laboratoires Expanscience
10, Av de l’Arche
92419 COURBEVOIE CEDEX
www.expanscience-ingredients.com
Maud CHAPIS François MONTCRIOL
Tel: +33 1 43 34 75 39 +33 1 43 34 75 81
Fax: +33 1 43 34 61 19 +33 1 43 34 61 19